Antibodies to the glycosphingolipid, sulfoglucuronyl paragloboside (SGPG) have been implicated in many different autoimmune diseases. For example, serum IgM antibodies to GM ganglioside have been found in patients with amyotrophic lateral sclerosis (ALS) (Younes-Chennoufi, B. A. et al., J. Neuroimmunol. 57:111-5 (1995)) chronic inflammatory demyelinating polyneuropathy (Yuki, N. et al., J. Neuroimmunol. 70:1-6 (1996)), and acute Guillain-Barre syndrome (Ilyas, AA. et al., J. Neurol. Sci. 105:108-17 (1991)).
Enzyme-linked immunosorbent assays (ELISA) have been used for identification of anti-glycosphingolipid antibodies; however, high background values frequently interfere with accurate assessment of the amount of such antibodies. Reliable measurement of anti-glycosphingolipid antibodies is critical for correct diagnosis of immune diseases.
The present invention pertains to methods of determining, in a test sample, the amount of antibodies directed against a specific nervous system antigen or antigens, using a modified solid-phase reactant. The method utilizes a solid-phase reactant, such as a microtiter plate, that is modified with carbonyl groups attached to its surface. One or more glycosphingolipids of interest (e.g., sulfoglucuronyl paragloboside (SGPG))are linked to the modified solid-phase reactant by an amide bond between an amino group of the glycosphingolipid and a carbonyl group attached to the solid-phase reactant. One or more control antigens, such as other glycosphingolipids, glycolipids, glycoproteins, gangliosides or carbohydrates, can also be attached on the surface of the modified solid-phase reactant. The modified solid-phase reactant having gycosphingolipid(s) of interest linked thereon is contacted with a test sample, such as a test sample of a bodily fluid (e.g., blood, serum, cerebrospinal fluid, or urine) from an individual, under conditions such that any antibody to the gycophingolipid(s) of interest that may be present in the test sample can bind to the glycosphingolipid(s) of interest linked to the modified solid-phase reactant. The amount of antibodies in the test sample to the glycosphingolipid(s) of interest is then determined using standard methods, such as enzyme-linked immunosorbent assay (ELISA) or another appropriate solid-phase assay. If a control antigen is attached on the modified solid-phase reactant, the level of antibodies in the test sample to the control antigen, can also be determined using the same methods. Specific reactivity of antibodies to the glycosphingolipid of interest is determined by the amount of antibody binding to the glycosphingolipid of interest that is above the amount of antibody binding to the control antigen.
The methods can be used in diagnosis of autoimmune diseases in an individual. The amount of antibody to a glycosphingolipid of interest in a test sample from the individual is determined using the methods. An amount of antibody to a glycosphingolipid of interest that is greater, by an amount that is statistically significant, than the amount of antibody to the glycosphingolipid of interest in a control sample, may be indicative of the presence of an autoimmune disease. Alternatively, an amount of antibody to a glycosphingolipid of interest that is equal to or greater than an established reference amount may be indicative of the presence of the disease. For example, the methods can be used as a preliminary screening assay; an amount of antibody to a glycosphingolipid of interest that is greater, by an amount that is statistically significant, than the amount of antibody to the glycosphingolipid of interest in a control sample, or an amount of antibody to a glycosphingolipid of interest that is equal to or greater than an established reference amount, is considered a xe2x80x9cpositivexe2x80x9d screening result that substantiates additional study of other antibodies that are involved in autoimmune disease.
The invention also pertains to test kits, containing modified solid-phase reactants, for use in the methods of the invention.
The high sensitivity and specificity of the methods can clarify the differential diagnosis of autoimmune diseases. Furthermore, a modified solid-phase reactant having carbonyl groups attached to its surface allows the use of a smaller amount of glycosphingolipid than the amount which would otherwise be necessary to perform similar assays with a solid-phase reactant not having this modification. In addition, a modified solid-phase reactant having carbonyl groups attached to its surface can be coated with a glycosphingolipid of interest without a need for toxic solvents; the coating is not affected by humidity, and yields consistent and reproducible assay results.